<p> Understanding enzyme catalysis is one of the major goals in biology. Ribonuclease A (RNase A) is a key system to understanding protein structure and function provides an attractive system to investigate the catalytic role of active site interactions. Crystal structures show a lysine residue (Lys41) situated in the RNase A active site, and mutagenesis studies suggest this residue is important for catalysis. To evaluate the catalytic importance of the Lys41-phosphate interaction, double mutant cycle analysis was used. Individual mutation of lysine to arginine (K41R) and substitution of a phosphate oxygen with sulfur led to &sim;350 and &sim;100-fold decrease in <i> k_cat/K</i>_M, respectively. However, in the K41R background, substitution of the same oxygen with sulfur decreased activity by a similar amount (within 2-fold) as it did with the wild-type enzyme. This result provides evidence that functional interaction between Lys41 and the phosphate backbone of RNA substrates may not be solely limited between the two groups.</p><p>

<p> <i>Aedes aegypti</i> mosquitoes can be found globally in tropical and subtropical urban areas and spread Zika, Dengue fever, yellow fever, and Chikungunya viruses. Current vector control methods are limited and nonspecific. The female <i>Ae. aegypti</i> mosquito uses blood meal proteins to obtain nutrients required for oogenesis; inhibition of the midgut trypsin-like serine proteases responsible for blood meal digestion may provide a novel method of vector control. <i>Ae. aegypti</i> blood meal digestion is complex and the role of uncharacterized serine proteases in blood digestion is unclear; specifically, a group of trypsin-like serine proteases (AaSPII&ndash;V) is expressed at constant levels before and following <i>Ae. aegypti</i> blood meal acquisition. This research focuses on the <i>in vitro</i> biochemical study of two specific <i>Ae. aegypti</i> trypsin-like serine proteases (AaSPII and AaSPIV) in order to gain further understanding of their role in blood meal digestion. The approach involved the successful cloning and bacterial expression of these soluble, recombinant proteases. Results from attempts to purify these proteases were unsuccessful but indicative of potential autocatalytic and autodigestive behavior. Future studies will focus on obtaining purified recombinant proteases for further study. The study of AaSPII and AaSPIV, as well as other midgut <i>Ae. aegypti</i> proteases, will aid in understanding the overall role proteases play in blood meal digestion and may eventually allow for the development of mosquito-specific enzyme inhibitors.</p><p>